To protect genomic integrity, humans are diploid, ie. they possess two copies of each chromosome (with the exception of the X- and the Y-chromosome that only exist in one copy), one from their father and one from their mother. If one gene on one chromosome is damaged, in many instances, the remaining second copy is sufficient to maintain gene function, thereby alleviating the otherwise deleterious effect. This fail-save mechanism that is essential for the survival of humans as a species turns out to be a nightmare for human geneticists: Whenever one copy of a gene is inactivated, the second copy usually buffers the effect, thereby masking the phenotype of that particular gene.
For the longest time, the only human cells that were known to be haploid were the gametes, i.e. the sperm cell and the oocyte. However, as these cells could not be propagated, they could not be used for genetic experiments (apart from ethical restrictions). More than 15 years ago, a group of scientists at Tufts University stumbled upon a cell line that had been isolated from a patient with chronic myelogenous leukemia (CML). That cell line, referred to as KBM-7 [1], was haploid for most chromosomes with the exception of chromosome 8 and a portion of chromosome 15, which were found to be disomic. Its near-haploid state could be maintained for several months in culture. Yet, eventually, KBM-7 cells convert to diploid [1], suggesting that the near-haploid karyotype rather represents a “meta-stable” state.
While the authors [1] noted that this cell line could be used “to facilitate the application of somatic cell genetics to the study of mammalian cell biology”, it took more than 10 years until Thijn Brummelkamp and his coworkers at the Whitehead Institute for Biomedical Research successfully applied KBM-7 cells for genetic experiments in human cells [2]. In this landmark paper, the authors used retroviral vectors to insert a conventional gene trap into the host genome and thereby disrupted gene expression at the site of integration. Most importantly, as retroviruses can be grown at very high titers, the method allowed the simultaneous disruptions of most non-essential human genes at very high coverage in a pool of mutant cells, thereby enabling unbiased positive selection screens [3]—similar to the screens done in yeast more than 15 years ago.
In addition to the unbiased genetics screens, the technology developed by Brummelkamp enabled the generation of a unique library of human cell lines in which every cell line bears one gene trap insertion at one defined position. Importantly, such libraries enable reverse genetics, ie. the study of individual mutants with regard to a specific phenotype under consideration. Such a library has recently been established. It contains almost 10,000 cell lines, covering more than 3,500 human genes [4]. In addition, this publication also contains a detailed genomic characterization of the parental KBM-7 cells by next generation sequencing and small nucleotide polymorphism (SNP) arrays. Based on these data, the disomic portion on chromosome 15 could be mapped to the region around chr15: 61,105,000-89,890,000.
While the genetic screens described above were very powerful, they were clearly limited by the availability of near-haploid human cell lines—at the time, KBM-7 was the only available cell line. Brummelkamp and coworkers therefore decided to reprogram KBM-7 cells to obtain induced pluripotent stem cells (iPSCs). While this turned out to be feasible, KBM-7-derived iPSCs lost their near-haploid karyotype during the de-differentiation procedure [5]. However, one by-product of this reaction was an adherent cell line called HAP1 that remained near-haploid and that showed a fibroblast-like morphology [6]. Importantly, HAP1 cells are not pluripotent, but they differ considerably from their KBM-7 parent cells in terms of growth, morphology and gene expression. Of note, HAP1 cells are also monosomic for chromosome 8 and are thus “more haploid” than their KBM-7 parents. However, HAP1 cells do retain the disomic fragment from chromosome 15 and can thus not be considered fully haploid. In addition, HAP1 cells are less stable than KBM-7 with regard to their near-haploid karyotype, i.e. they convert to the diploid state more readily than KBM-7 cells.
Bacteria have a need to maintain their genomic integrity and defend against invading viruses and plasmids. Recently, genomic loci with clustered, regularly interspaced, short palindromic repeats (CRISPRs) were found in bacteria and were shown to mediate adaptive immunity to invading phages [7]: Bacteria can capture short nucleic acid sequences from the phage and integrate them in the CRISPR loci. Small RNAs, produced by transcription of the CRISPR loci, can guide a set of bacterial endonucleases to cleave the genomes of invading pathogens.
The minimal requirements for one bacterial endonuclease, CAS9 from Streptococcus pyogenes, were characterized by purifying the enzyme and reconstituting the cleavage reaction in vitro [8]. Surprisingly, CAS9 itself is sufficient for endonuclease cleavage and no further polypeptides are required for the cleavage reaction. In addition, CAS9 requires two RNA cofactors: a constant tracrRNA and a variable crRNA. Importantly, the crRNA can be used to reprogram the cleavage specificity of CAS9, thereby enabling the targeting of CAS9 to genomic loci of interest. Cleavage specificity is limited by the protospacer adjacent motif (PAM) that is specific to CAS9 and lies adjacent to the cleavage site. In an attempt to simplify the system, crRNA and tracrRNA were fused to give rise to one chimeric RNA molecule referred to as the guide RNA.
US 2010/0076057 A1 discloses the target DNA interference with crRNA and CRISPR-associated (cas) proteins, in particular for horizontal gene transfer based on the use of CRISPR sequences.
The RNA-directed DNA cleavage by the CAS9-crRNA complex is described by WO 2013/141680 A1 and WO 2013/142578 A1.
It is the object of the present invention to engineer near-haploid cells to reduce diploidy of the cells. It is the further object to provide a cell line of haploid karyotype that is karyotypically stable.